The principal research goal of the Synaptic Function Unit is to understand the molecular mechanisms underlying the neurotransmitter release process and its modulation. In past year, we have obtained promising results on the following four projects: (1) Continued characterization of a novel SNARE-associated protein called Snapin, which was isolated in our laboratory in 1998. Snapin is a synaptic vesicle membrane associated protein involved in neurotransmission by directly binding to SNAP-25 and potentiating interactions between the SNARE proteins and synaptotagmin, a key molecular intermediate in Ca2+-dependent exocytosis. Introducing truncated Snapin and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons (SCGNs) in culture reversibly inhibits synaptic transmission. We also identified that several residues on Snapins carboxyl terminal are critical for its binding to SNAP-25. The paper describing our work on cloning and biochemical and physiological characterization of Snapin was published in Nature Neuroscience (2, 119-124, 1999). (2) More recently, we have found that Snapin may be a target for cAMP-dependent protein kinase A (PKA) in the neurotransmitter secretory machinery. We found that both recombinant and native Snapin derived from rat brain synaptosomes are phosphorylated by PKA. Deletion mutation and PCR-based site-directed mutagenetic experiments pinpointed the phosphorylation site to serine50. PKA-phosphorylation of Snapin significantly increases its binding to SNAP-25. A Snapin mutation of serine50 to aspartic acid (S50D) mimics the effect of PKA phosphorylation of Snapin, suggesting that the introduction of a negatively charged residue at serine50 is critical to regulate Snapin?s function. Furthermore, the Snapin S50D mutant enhances the association of synaptotagmin with the SNARE complex. Our results suggest that Snapin is a PKA target for modulating transmitter release and neuronal plasticity via the cAMP-dependent signal transduction pathway. The paper on PKA phosphorylation of Snapin has been submitted. (3) We have cloned and characterized a protein called syntaphilin that interacts with syntaxin-1A. Northern, immunoblotting, and immunocytochemical analyses demonstrated that syntaphilin is selectively expressed in brain and is highly enriched at nerve terminals. In vitro binding and co-immunoprecipitation studies revealed that syntaphilin competes with SNAP-25 for binding to syntaxin-1, and inhibits SNARE complex formation by absorbing free syntaxin-1 at nerve terminals. Transient over-expression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls the amount of free syntaxin-1 available for SNARE complex assembly, and thereby regulates synaptic vesicle exocytosis. Our paper describing the cloning and characterization of syntaphilin is in press at Neuron. (4) Using the yeast two-hybrid system we have isolated another syntaxin-1A binding protein from the human brain cDNA library. The deduced amino acid sequence is identical to a recently cloned gene called SNAP-29. Using the 3-untranslated cDNA fragment as a probe, our Northern blots showed that a major species of mRNA, 4.4 kb in length, was detected in multiple tissues including brain. The specific interaction of SNAP-29 with syntaxin-1A was further confirmed using in vitro binding studies with recombinant proteins and co-immunoprecipitation analysis with co-transfected HEK 293 T cell lysates. The amino terminal half of SNAP-29, which contains a coiled coil domain, is necessary and sufficient for its interaction with syntaxin-1A. Furthermore, SNAP-29 competes with native SNAP-25 for binding to syntaxin-1 and thus inhibits SNARE complex formation at nerve terminals. Introduction of the amino terminal half of SNAP-29 into presynaptic superior cervical ganglion neurons (SCGNs) in culture significantly inhibits synaptic transmission. Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a cytoplasmic regulator for the assembly of the SNARE complex and participate in the docking/fusion process of synaptic vesicle exocytosis. The manuscript describing our work on SNAP-29 is in preparation.